The overall aim of the proposed research is to characterize the signal regulation and endocytic sorting of protease-activated receptor-4 (PAR4), the most understudied and underappreciated of the three thrombin receptors. Thrombin is a serine protease mediates hemostasis, thrombosis, and inflammatory responses to vascular injury and contributes to the pathogenesis of cardiovascular disease. Signaling by thrombin is implicated in progression and metastasis of certain types of malignant cancers. The majority of thrombin's physiological actions occur through activation of protease-activated receptors (PARs), which are a class of heptahelical transmembrane GPCRs that are irreversibly activated by proteolytic cleavage of the extracellular N-terminus. PAR1 and PAR4 both signal in response to cleavage by thrombin, but PAR1 has a higher affinity for thrombin, which initially led many to believe that PAR4 simply played a redundant role in thrombin signaling. As a result, the majority of PAR-directed therapies currently in development are focused on regulation of PAR1. However, recent studies have provided strong evidence suggesting that PAR4 signaling plays a necessary role distinct from that of PAR1. Furthermore, several cell types, such as human platelets and lung endothelial cells, express both PAR1 and PAR4. Therefore, it is essential that PAR4 be taken into account in the development of therapeutics aimed at regulating thrombin signaling. Because PARs are irreversibly activated, the mechanisms that regulate PAR signaling are critical for the fidelity of thrombin signaling. PAR1 signal regulation is well-characterized, and mediated by post-translational modifications, interactions with adaptor proteins, clathrin-mediated endocytosis, and eventual lysosomal degradation. However, virtually nothing is known about the signal regulation and sorting of activated PAR4. Thus, one aim of the proposed research is to delineate the mechanisms that regulate PAR4's signaling and trafficking. In platelets, PAR1 signaling has been shown to be responsible for the initial stages of clot development while PAR4 is important in the later stages of the process, aiding in clot stabilization. Certain endothelial cells also co-express PAR4 with PAR1. In this context PAR1 signaling is known to induce changes in endothelial barrier permeability by a well-defined pathway. The role of PAR4 in this process is completely uncharacterized. Thus, I will examine PAR4's contribution to thrombin-stimulated endothelial cell responses using primary human lung endothelial cells as a model system. There is limited evidence in the literature that suggests PAR4 forms heterodimers with P2Y12. I will address this question using biochemical approaches and sophisticated imaging techniques in order provide more definitive evidence for the existence of purported PAR4/P2Y12 heterodimers and the impact they have on trafficking and G-protein selectivity.